Project Summary/Abstract The unicellular parasite Trichomonas vaginalis is responsible for the most prevalent, non-viral, sexually- transmitted infection worldwide, with approximately billion people contracting trichomoniasis annually. Trichomoniasis is the most common parasitic infection in the US, with an annual incidence estimated at ~5 million cases. Nevertheless, this parasite is vastly understudied. As such, T. vaginalis was classified by the Center of Disease Control and Prevention as a neglected infection in the US in 2014. In addition to being a common cause of vaginitis, trichomoniasis is associated with adverse inflammatory sequelae, that can contribute to pregnancy complications and neonatal mortality, the spread of HIV, and increased metastasis of urogenital cancers. The incidence of infection, the growing recognition that T. vaginalis is associated with long-term health consequences and an increase in the number of drug resistant clinical isolates of T. vaginalis underscore the need to develop new chemotherapeutic and vaccine design strategies. A much better understanding of processes involved in infectivity and pathogenesis, such as those proposed here, will be imperative to achieve these goals. Several years ago we discovered that T. vaginalis secretes small, membrane-bound extracellular vesicles (EVs), that mediate host:parasite interactions. We have shown that parasite proteins are transferred to host cells via EVs, which in turn, modulates both parasite adherence to host cells and the host cell responses. Host:pathogen cross- talk mediated by EVs likely contributes to parasite colonization of the host and down-regulation of cytokines that elicit immune cells to the site of infection. This proposal focuses on the molecular and cellular mechanisms used for host cell internalization of EVs: a process required for EV-mediated communication between the parasite and host. We will also examine the effect of EVs on the survival of parasites in vivo using a newly-developed mouse model. To this end, we propose to: characterize biochemical properties of an EV ligand (Tv 4-alpha- glucanotransferase) that binds host cell heparan sulfate proteoglycans and drives EV internalization by host cells (Aim 1); isolate and identify EV receptor(s) on the host cell and test whether the receptor(s) are required for EV internalization (Aim 2) and to identify EV proteins involved in host cell internalization and test whether EVs affect parasite survival during early stage infection in vivo using isogenic T. vaginalis strains that differ in host cell adherence (Aim 3). We will uncover biochemical and cellular mechanisms that promote parasite infection and will reveal whether our in vitro findings supporting a role for EVs in host cell colonization are confirmed in vivo. Novel mechanisms are likely to be found, as very little mechanistic data on the interaction or uptake of any parasite-derived EV with host cells have been reported. In addition to expanding our knowledge of host:pathogen interactions, these studies will increase our overall knowledge of how EVs mediate cell:cell communication and will contribute to a better understanding of the role of EVs in infectious diseases. These findings could also enable future development of new therapeutic approaches.